2-Methylene-19,26,27-Trinor-(20S)-1Alpha-Hydroxyvitamin D3 and Its Uses

ABSTRACT

This invention discloses 2-methylene-19,26,27-trinor-(20S)-vitamin D analogs, and specifically 2-methylene-19,26,27-trinor-(20S)-1α-hydroxyvitamin D 3  and pharmaceutical uses therefor. This compound exhibits pronounced activity in arresting the proliferation of undifferentiated cells and inducing their differentiation to the monocyte thus evidencing use as an anti-cancer agent and for the treatment of skin diseases such as psoriasis as well as skin conditions such as wrinkles, slack skin, dry skin and insufficient sebum secretion. This compound also has little, if any, calcemic activity and therefore may be used to treat autoimmune disorders or inflammatory diseases in humans as well as renal osteodystrophy. This compound may also be used for the treatment or prevention of obesity.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.60/629,965, filed Nov. 22, 2004.

BACKGROUND OF THE INVENTION

This invention relates to vitamin D compounds, and more particularly to2-methylene-19,26,27-trinor-(20S)-1α-Hydroxyvitamin D₃ and itspharmaceutical uses.

The natural hormone, 1α,25-dihydroxyvitamin D₃ and its analog inergosterol series, i.e. 1α,25-dihydroxyvitamin D₂ are known to be highlypotent regulators of calcium homeostasis in animals and humans, andtheir activity in cellular differentiation has also been established,Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Manystructural analogs of these metabolites have been prepared and tested,including 1α-hydroxyvitamin D₃, 1α-hydroxyvitamin D₂, various side chainhomologated vitamins and fluorinated analogs. Some of these compoundsexhibit an interesting separation of activities in cell differentiationand calcium regulation. This difference in activity may be useful in thetreatment of a variety of diseases such as renal osteodystrophy, vitaminD-resistant rickets, osteoporosis, psoriasis, and certain malignancies.

Another class of vitamin D analogs, i.e. the so called 19-nor-vitamin Dcompounds, is characterized by the replacement of the A-ring exocyclicmethylene group (carbon 19), typical of the vitamin D system, by twohydrogen atoms. Biological testing of such 19-nor-analogs (e.g., 1α,25-dihydroxy-19-nor-vitamin D₃) revealed a selective activity profilewith high potency in inducing cellular differentiation, and very lowcalcium mobilizing activity. Thus, these compounds are potentiallyuseful as therapeutic agents for the treatment of malignancies, or thetreatment of various skin disorders. Two different methods of synthesisof such 19-nor-vitamin D analogs have been described (Perlman et al.,Tetrahedron Lett. 31, 1823 (1990); Perlman et al., Tetrahedron Lett. 32,7663 (1991), and DeLuca et al., U.S. Pat. No. 5,086,191).

In U.S. Pat. No. 4,666,634, 2β-hydroxy and alkoxy (e.g., ED-71) analogsof 1α,25-dihydroxyvitamin D₃ have been described and examined by Chugaigroup as potential drugs for osteoporosis and as antitumor agents. Seealso Okano et al., Biochem. Biophys. Res. Commun. 163, 1444 (1989).Other 2-substituted (with hydroxyalkyl, e.g., ED-120, and fluoroalkylgroups) A-ring analogs of 1α,25-dihydroxyvitamin D₃ have also beenprepared and tested (Miyamoto et al., Chem. Pharm. Bull. 41, 1111(1993); Nishii et al., Osteoporosis Int. Suppl. 1, 190 (1993); Posner etal., J. Org. Chem. 59, 7855 (1994), and J. Org. Chem. 60, 4617 (1995)).

2-substituted analogs of 1α,25-dihydroxy-19-nor-vitamin D₃ have alsobeen synthesized, i.e. compounds substituted at 2-position with hydroxyor alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713), with 2-alkylgroups (DeLuca et al U.S. Pat. No. 5,945,410), and with 2-alkylidenegroups (DeLuca et al U.S. Pat. No. 5,843,928), which exhibit interestingand selective activity profiles. All these studies indicate that bindingsites in vitamin D receptors can accommodate different substituents atC-2 in the synthesized vitamin D analogs.

In a continuing effort to explore the 19-nor class of pharmacologicallyimportant vitamin D compounds, analogs which are characterized by thepresence of a methylene substituent at carbon 2 (C-2), a hydroxyl groupat carbon 1 (C-1), and a shortened side chain attached to carbon 20(C-20) have also been synthesized and tested.1α-hydroxy-2-methylene-19-nor-pregnacalciferol is described in U.S. Pat.No. 6,566,352 while 1α-hydroxy-2-methylene-19-nor-homopregnacalciferolis described in U.S. Pat. No. 6,579,861 and1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol is described inU.S. Pat. No. 6,627,622. All three of these compounds have relativelyhigh binding activity to vitamin D receptors and relatively high celldifferentiation activity, but little if any calcemic activity ascompared to 1α,25-dihydroxyvitamin D₃. Their biological activities makethese compounds excellent candidates for a variety of pharmaceuticaluses, as set forth in the '352, '861 and '622 patents.

SUMMARY OF THE INVENTION

The present invention is directed toward2-methylene-19,26,27-trinor-(20S)-vitamin D analogs, and morespecifically toward 2-methylene-19,26,27-trinor-(20S)-1α-hydroxyvitaminD₃, their biological activity, and various pharmaceutical uses for thesecompounds.

Structurally these analogs are characterized by the general formula Ishown below:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group. The preferred analog is2-methylene-19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃ which has thefollowing formula Ia:

The above compounds of formula I, especially the compound of formula Ia,exhibit a desired, and highly advantageous, pattern of biologicalactivity. These compounds are characterized by relatively high bindingto vitamin D receptors, but very low intestinal calcium transportactivity, as compared to that of 1α, 25-dihydroxyvitamin D₃, and havevery low ability to mobilize calcium from bone, as compared to1α,25-dihydroxyvitamin D₃. Hence, these compounds can be characterizedas having little, if any, calcemic activity. It is undesirable to raiseserum calcium to supraphysiologic levels when suppressing thepreproparathyroid hormone gene (Darwish & DeLuca, Arch. Biochem.Biophys. 365, 123-130, 1999) and parathyroid gland proliferation. Theseanalogs having little or no calcemic activity while very active ondifferentiation are expected to be useful as a therapy for suppressionof secondary hyperparathyroidism of renal osteodystrophy.

The compounds I, and particularly Ia, of the invention have also beendiscovered to be especially suited for treatment and prophylaxis ofhuman disorders which are characterized by an imbalance in the immunesystem, e.g. in autoimmune diseases, including multiple sclerosis,lupus, diabetes mellitus, host versus graft rejection, and rejection oforgan transplants; and additionally for the treatment of inflammatorydiseases, such as rheumatoid arthritis, asthma, and inflammatory boweldiseases such as celiac disease, ulcerative colitis and Crohn's disease.Acne, alopecia and hypertension are other conditions which may betreated with the compounds of the invention.

The above compounds I, and particularly Ia, are also characterized byrelatively high cell differentiation activity. Thus, these compoundsalso provide a therapeutic agent for the treatment of psoriasis, or asan anti-cancer agent, especially against leukemia, colon cancer, breastcancer, skin cancer and prostate cancer. In addition, due to theirrelatively high cell differentiation activity, these compounds provide atherapeutic agent for the treatment of various skin conditions includingwrinkles, lack of adequate dermal hydration, i.e. dry skin, lack ofadequate skin firmness, i.e. slack skin, and insufficient sebumsecretion. Use of these compounds thus not only result in moisturizingof skin but also improves the barrier function of skin.

The compounds of the invention of formula I, and particularly formulaIa, are also useful in preventing or treating obesity, inhibitingadipocyte differentiation, inhibiting SCD-1 gene transcription, and/orreducing body fat in animal subjects. Therefore, in some embodiments, amethod of preventing or treating obesity, inhibiting adipocytedifferentiation, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal subject includes administering to the animalsubject, an effective amount of one or more of the compounds or apharmaceutical composition that includes one or more of the compounds offormula I. Administration of one or more of the compounds or thepharmaceutical compositions to the subject inhibits adipocytedifferentiation, inhibits gene transcription, and/or reduces body fat inthe animal subject.

One or more of the compounds may be present in a composition to treatthe above-noted diseases and disorders in an amount from about 0.01μg/gm to about 1000 μg/gm of the composition, preferably from about 0.1μg/gm to about 500 μg/gm of the composition, and may be administeredtopically, transdermally, orally, rectally, nasally, sublingually orparenterally in dosages of from about 0.01 μg/day to about 1000 μg/day,preferably from about 0.1 μg/day to about 500 μg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-6 illustrate various biological activities of2-methylene-19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃, hereinafterreferred to as “OM,” as compared to the native hormone 1α,25-dihydroxyvitamin D₃, hereinafter “1,25(OH)₂D₃.”

FIG. 1 is a graph illustrating the relative activity of OM and1,25(OH)₂D₃ to compete for binding with [³H]-1,25-(OH)₂-D₃ to thefull-length recombinant rat vitamin D receptor;

FIG. 2 is a graph illustrating the percent HL-60 cell differentiation asa function of the concentration of OM and 1,25(OH)₂D₃;

FIG. 3 is a bar graph illustrating the in vitro transcription activityof 1,25(OH)₂D₃ as compared to OM;

FIGS. 4 and 5 are bar graphs illustrating the bone calcium mobilizationactivity of 1,25(OH)₂D₃ as compared to OM. Each graph represents aseparate batch of vitamin D-deficient animals. The results depicted inFIG. 5 are not vehicle-controlled, but rather each animal serves as itsown control because the animals were bled pre- and post-dose; and

FIG. 6 is a bar graph illustrating the intestinal calcium transportactivity of 1,25(OH)₂D₃ as compared to OM.

DETAILED DESCRIPTION OF THE INVENTION

2-methylene-19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃ (referred toherein as OM) was synthesized and tested. Structurally, this 19-noranalog and its pro-drug (in protected hydroxy form) is characterized bythe general formula I previously illustrated herein, while the analog OMitself is represented by the formula Ia.

The preparation of 2-methylene-19,26,27-trinor-(20S)-1α-hydroxyvitaminD₃ having the structure Ia as well as compounds I can be accomplished bya common general method, i.e. the condensation of a bicyclicWindaus-Grundmann type ketone II with the allylic phosphine oxide III tothe corresponding 2-methylene-19,26,27-trinor-vitamin D analog IVfollowed by deprotection at C-1 and C-3 in the latter compound:

In the structures III and IV, groups X₁ and X₂ are hydroxy-protectinggroups, preferably t-butyldimethylsilyl, it being also understood thatany functionalities that might be sensitive, or that interfere with thecondensation reaction, be suitably protected as is well-known in theart. The process shown above represents an application of the convergentsynthesis concept, which has been applied effectively for thepreparation of vitamin D compounds [e.g. Lythgoe et al., J. Chem. Soc.Perkin Trans. I, 590 (1978); Lythgoe, Chem. Soc. Rev. 9, 449 (1983); Tohet al., J. Org. Chem. 48, 1414 (1983); Baggiolini et al., J. Org. Chem.51, 3098 (1986); Sardina et al., J. Org. Chem. 51, 1264 (1986); J. Org.Chem. 51, 1269 (1986); DeLuca et al., U.S. Pat. No. 5,086,191; DeLuca etal., U.S. Pat. No. 5,536,713].

The hydrindanone of the general structure II is not known. It can beprepared by the method shown on Schemes 1 and 2 (see the preparation ofcompound OM).

For the preparation of the required phosphine oxides of generalstructure III, a synthetic route has been developed starting from amethyl quinicate derivative which is easily obtained from commercial(1R,3R,4S,5R)-(−)-quinic acid as described by Perlman et al.,Tetrahedron Lett. 32, 7663 (1991) and DeLuca et al., U.S. Pat. No.5,086,191.

The overall process of the synthesis of compounds I and Ia isillustrated and described more completely in U.S. Pat. No. 5,843,928entitled “2-Alkylidene-19-Nor-Vitamin D Compounds” the specification ofwhich is specifically incorporated herein by reference.

As used in the description and in the claims, the term“hydroxy-protecting group” signifies any group commonly used for thetemporary protection of hydroxy functions, such as for example,alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafterreferred to simply as “silyl” groups), and alkoxyalkyl groups.Alkoxycarbonyl protecting groups are alkyl-O—CO— groupings such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl,benzyloxycarbonyl or allyloxycarbonyl. The term “acyl” signifies analkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or acarboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, malonyl,succinyl, glutaryl group, or an aromatic acyl group such as benzoyl, ora halo, nitro or alkyl substituted benzoyl group. The word “alkyl” asused in the description or the claims, denotes a straight-chain orbranched alkyl radical of 1 to 10 carbons, in all its isomeric forms.Alkoxyalkyl protecting groups are groupings such as methoxymethyl,ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl andtetrahydropyranyl. Preferred silyl-protecting groups are trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, dibutylmethylsilyl,diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t-butylsilyl andanalogous alkylated silyl radicals. The term “aryl” specifies a phenyl-,or an alkyl-, nitro- or halo-substituted phenyl group.

A “protected hydroxy” group is a hydroxy group derivatised or protectedby any of the above groups commonly used for the temporary or permanentprotection of hydroxy functions, e.g. the silyl, alkoxyalkyl, acyl oralkoxycarbonyl groups, as previously defined. The terms “hydroxyalkyl”,“deuteroalkyl” and “fluoroalkyl” refer to an alkyl radical substitutedby one or more hydroxy, deuterium or fluoro groups respectively.

More specifically, reference should be made to the following descriptionas well as to Schemes 1, 2 and 3 herein for a detailed illustration ofthe preparation of compound OM.

Preparation of(20S)-de-A,B-8β-(tert-butyldimethylsilyl)oxy-20-(hydroxymethyl)pregnane(2)

Ozone was passed through a solution of vitamin D₂ (3 g, 7.6 mmol) inmethanol (250 mL) and pyridine (2.44 g, 2.5 mL, 31 mmol) for 50 min at−78° C. The reaction mixture was then flushed with an oxygen for 15 minto remove the residual ozone and the solution was treated with NaBH₄(0.75 g, 20 mmol). After 20 min the second portion of NaBH₄ (0.75 g, 20mmol) was added and the mixture was allowed to warm to room temperature.The third portion of NaBH₄ (0.75 g, 20 mmol) was then added and thereaction mixture was stirred for 18 h. The reaction was quenched withwater (40 mL) and the solution was concentrated under reduced pressure.The residue was extracted with ethyl acetate (3×80 mL) and the combinedorganic phase washed with 1M aq. HCl, saturated aq. NaHCO₃, dried(Na₂SO₄) and concentrated under reduced pressure. The residue waschromatographed on silica gel with hexane/ethyl acetate (75:25) to give(20S)-de-A,B-20-(hydroxymethyl)pregnan-8β-ol 1 (1.21 g, 75% yield) aswhite crystals.

tert-Butyldimethylsilyl trifluoromethanesulfonate (3.24 mL, 3.72 g, 14.1mmol) was added to a solution of the 8β,20-diol 1 (1 g, 4.7 mmol) and2,6-lutidine (1.64 mL, 1.51 g, 14.1 mmol) in anhydrous DMF (15 mL) at 0°C. The mixture was stirred under argon at 0° C. for 1 h and then at roomtemperature for 18 h. The reaction was quenched with water (50 mL) andextracted with ethyl acetate (3×30 mL). The combined organic phasewashed with brine, dried (Na₂SO₄) and concentrated under reducedpressure. The residue was dissolved in anhydrous THF (8 mL),triethylamine (3 mL, 2.17 g, 21.5 mmol) and a solution oftetrabutylammonium fluoride (1 M in THF, 6.5 mL, 6.5 mmol) were added,followed by freshly activated molecular sieves 4A (3 g). The reactionmixture was stirred under argon at room temperature for 4 h, thenfiltered through a short layer of Celite and evaporated. The residue wasdissolved in ethyl acetate (30 mL), washed with brine, water, dried(Na₂SO₄) and concentrated under reduced pressure. The pure alcohol 2(1.42 g, 93% yield) was isolated by a chromatography on silica gel withhexane/ethyl acetate (97.5:2.5→95:5), as a colorless oil: ¹H NMR (500MHz, CDCl₃) δ 4.00 (1H, d, J=2.4 Hz, 8α-H), 3.63 (1H, dd, J=10.5, 3.2Hz, 22-H), 3.39 (1H, dd, J=10.5, 6.8 Hz, 22-H), 1.94 (1H, br.d, J=12.5Hz), 1.02 (3H, d, J=6.6 Hz, 21-H₃), 0.924 (3H, s, 18-H₃), 0.882 (9H, s,Si-t-Bu), 0.005 and −0.010 (each 3H, each s, each Si—Me); ¹³C NMR (125MHz) δ 69.29 (d, C-8), 67.94 (t, C-22), 53.06 (d), 52.80 (d), 42.12 (s,C-13), 40.54 (t), 38.27 (d), 34.39 (t), 26.79 (t), 25.79 (q, SiCMe₃),23.08 (t), 18.00 (s, SiCMe₃), 17.61 (t), 16.65 (q, C-21), 13.75 (q,C-18), −4.81 and −5.18 (each q, each SiMe).

Preparation of(20S)-de-A,B-8β-(tert-butyldimethylsilyl)oxy-20-formylpregnane (3)

Sulfur trioxide pyridine complex (1.32 g, 8.28 mmol) was added to asolution of the alcohol 2 (451 mg, 1.38 mmol), triethylamine (960 μL,697 mg, 6.9 mmol) in anhydrous methylene chloride (20 mL) and anhydrousDMSO (5 mL) at 0° C. The reaction mixture was stirred under argon at 0°C. for 20 min. and then concentrated. The residue was purified by columnchromatography on silica gel with hexane/ethyl acetate (95:5) to givethe aldehyde 3 (364 mg, 81% yield) as an oil: ¹H NMR (500 MHz, CDCl₃) δ9.55 (1H, d, J=3.1 Hz, CHO), 4.00 (1H, S, 8α-H), 2.33 (1H, m, 20-H),1.89 (1H, dm, J=12.4 Hz), 1.07 (3H, d, J=6.8 Hz, 21-H₃), 0.939 (3H, s,18-H₃), 0.862 (9H, s, Si-t-Bu), −0.009 and −0.026 (each 3H, each s, eachSiMe); ¹³C NMR (125 MHz) δ 205.37 (d, CHO), 68.99 (d, C-8), 52.28 (d),51.58 (d), 49.15 (d), 42.58 (s, C-13), 40.35 (t), 34.29 (t), 26.16 (t),25.74 (q, SiCMe₃), 23.27 (t), 17.96 (s, SiCMe₃), 17.52 (t), 14.04 (q,C-21), 13.28 (q, C-18), −4.85 and −5.23 (each q, each SiMe).

Preparation of(20R)-de-A,B-8β-(tert-butyldimethylsilyl)oxy-20-(hydroxymethyl)pregnane(4)

The aldehyde 3 (364 mg, 1.12 mmol) was dissolved in methylene chloride(15 mL) and a 40% aq. n-Bu₄NOH solution (1.47 mL, 1.45 g, 2.24 mmol) wasadded. The resulting mixture was stirred under argon at room temperaturefor 16 h, diluted with methylene chloride (20 mL), washed with water,dried (Na₂SO₄) and concentrated under reduced pressure. A residue waschromatographed on silica gel with hexane/ethyl acetate (95:5) to afforda mixture of aldehyde 3 and its 20-epimer (292 mg, 80% yield) in ca. 1:2ratio (by ¹H NMR).

This mixture of aldehydes (292 mg, 0.9 mmol) was dissolved in THF (5 mL)and NaBH (64 mg, 1.7 mmol) was added, followed by a dropwise addition ofethanol (5 mL). The reaction mixture was stirred at room temperature for30 min and it was quenched with a saturated aq. NH₄Cl solution. Themixture was extracted with ether (3×20=mL) and the combined organicphase washed with water, dried (Na₂SO₄) and concentrated under reducedpressure. The residue was chromatographed on silica gel withhexane/ethyl acetate (96:4→80:20) to give the desired, pure(20R)-alcohol 4 (160 mg, 55% yield) as an oil and a mixture of 4 and its20-epimer 2 (126 mg, 43% yield) in ca. 1:3 ratio (by ¹H NMR).

4: [α]_(D)+40.8° (c 1.09, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 4.00 (1H, d,J=1.9 Hz, 8α-H), 3.70 (1H, dd, J=10.6, 3.2 Hz, 22-H), 3.43 (1H, dd,J=10.6, 7.0 Hz, 22-H), 0.94 (3H, d, J=6.7 Hz, 21-H₃), 0.927 (3H, s,18-H₃), 0.884 (9H, s, Si-t-Bu), 0.007 and −0.006 (each 3H, each s,SiMe₂); ¹³C NMR (125 MHz) δ 69.30 (d, C-8), 66.83 (t, C-22), 53.02 (d),52.96 (d), 41.91 (s, C-13), 40.12 (t), 37.48 (d), 34.38 (t), 26.71 (t),25.79 (q, SiCMe₃), 22.85 (t), 18.01 (s, SiCMe₃), 17.64 (t), 16.58 (q,C-21), 14.07 (q, C-18), 4.81 and −5.18 (each q, each SiMe).

Preparation of(20R)-de-A,B-8β-(tert-butyldimethylsilyl)oxy-20-(iodomethyl)pregnane (5)

A solution of iodine (471 mg, 1.84 mmol) in methylene chloride (30 mL)was slowly added to a solution of triphenylphosphine (482 mg, 1.84 mmol)and imidazole (250 mg, 3.68 mmol) in methylene chloride (15 mL) at 0° C.After 15 min. a solution of alcohol 4 (149 mg, 0.46 mmol) in methylenechloride (3 mL) was added into the mixture. After being stirring for 20min. at 0° C., followed by 18 h at room temperature, the reactionmixture washed with water, dried (Na₂SO₄) and concentrated under reducedpressure. The residue was chromatographed on silica gel withhexane/ethyl acetate (97:3) to give the desired iodide 5 (201 mg, 100%):[α]_(D)−0.3° (c 0.97, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 3.99 (1H, s,8α-H), 3.46 (1H, dd, J=9.5, 2.9 Hz, 22-H), 3.18 (1H, dd, J=9.5, 6.4 Hz),1.88-1.74 (3H, m), 1.67 (1H, dm, J=13.9 Hz), 0.95 (3H, d, J=6.4 Hz,21-H₃), 0.918 (3H, s, 18-H₃), 0.882 (9H, s, Si-t-Bu), 0.008 and −0.008(each, 3H, each s, SiMe₂); ¹³C NMR (125 MHz) δ 69.27 (d, C-8), 55.19(d), 52.69 (d), 41.99: (s, C-13), 40.48 (t), 36.15 (d), 34.24 (t), 26.90(t), 25.80 (q, SiCMe₃), 22.81 (t), 21.38 (q, C-21), 19.58 (t), 18.02 (s,SiCMe₃), 17.63 (t), 14.12 (q, C-18), −4.79 and −5.17 (each q, eachSiMe); MS (EI) m/z 436 (15, M⁺), 421 (8, M⁺-CH₃), 393 (9, M⁺-C₃H₇), 379(98, M⁺-t-Bu), 303 (65, M⁺-t-BuMe₂SiOH—H), 177 (70), 135 (70), 95 (55),75 (100); exact mass calculated for C₁₉H₃₇OSiI (M⁺) 436.1658, found436.1672.

Preparation of(20S)-de-A,B-8β-(tert-butyldimethylsilyl)oxy-20-(3-isopropoxycarbonyl)propyl-pregnane(6)

A mixture of zinc powder (124 mg, 1.9 mmol), anhydrous pyridine (4 mL)and isopropyl acrylate (235 μL, 217 mg, 1.9 mmol) was warmed to 50° C.,then nickel(II) chloride hexahydrate (109 mg, 0.46 mmol) was added. Theresulting mixture was warmed to 65° C. and stirred for 2 h until itsgreen color turned to reddish brown one. After cooling to 0° C., asolution of iodide 5 (222 mg, 0.51 mmol) in anhydrous pyridine (3 mL)was added and the reaction mixture was stirred for 4 h at roomtemperature. The mixture was diluted with ethyl acetate (20 mL) and theresulting precipitate was filtered off through a pad of Celite. Thefiltrate washed with 5% aq. HCl and brine, dried (Na₂SO₄) andconcentrated under reduced pressure. The residue was chromatographed onsilica gel with hexane and hexane/ethyl acetate (95:5) to give the ester6 (177 mg, 82%): [α]_(D)+19.7° (c 1.13, CHCl₃); ¹H NMR (400 MHz, CDCl₃)δ 5.00 (1H, sep, J=6.3 Hz, OCHMe₂), 3.99 (1H, d, J=2.2 Hz, 8α-H), 2.23(1H, dd, J=7.4, 2.5 Hz, 24-H), 2.21 (1H, dd, J=6.8, 1.9 Hz, 24-H), 1.90(1H, dm, J=12.2 Hz), 1.22 (6H, d, J=6.3 Hz, OCHMe₂), 0.895 (3H, s,18-H₃), 0.881 (9H, s, Si-t-Bu), 0.82 (3H, d, J=6.6 Hz, 21-H₃), 0.001 and−0.012 (each, 3H, each s, SiMe₂); ¹³C NMR (100 MHz) δ 173.48 (s,COO-iPr), 69.45 (d, C-8), 67.31 (d, COOCHMe₂), 56.29 (d), 53.08 (d),42.16 (s, C-13), 40.64 (t), 35.05 (t), 34.71 (t), 34.51 (d), 34.44 (t),27.16 (t), 25.80 (q, SiCMe₃), 22.93 (t), 21.92 (t), 21.86 (q, COOCHMe₂),18.48 (q, C-21), 18.02 (t), 17.69 (s, SiCMe₃), 14.01 (q, C-18), −4.79and −5.16 (each q, each SiMe); MS (EI) m/z 424 (5, M⁺), 409 (15,M⁺-CH₃), 381 (35, M⁺-C₃H₇), 367 (89, M⁺-t-Bu), 321 (39,M⁺-CH₃COOCHMe₂-H), 307 (85, M⁺-CH₃CH₂COOCHMe₂-H), 283 (65), 265 (41),249 (45), 233 (60), 215 (73), 189 (70), 163 (78), 135 (86), 109 (70), 95(79), 75 (100); exact mass calculated for C₂₅H₄₈O₃Si (M⁺) 424.3373,found 424.3371.

Preparation of(20S)-de-A,B-8β-(tert-butyldimethylsilyl)oxy-20-(4-hydroxy)butyl-pregnane(7)

Lithium aluminium hydride (20 mg, 0.53 mmol) was added to a solution ofester 6 (188 mg, 0.28 mmol) in anhydrous THF (5 mL) at 0° C. Thereaction mixture was stirred for 30 min at 0° C., then a cooling bathwas removed and the stirring was continued for additional 19 h at roomtemperature. The excess hydride was quenched by careful, successiveaddition of sat. aq. NH₄Cl. Methylene chloride (15 mL) and Celite (0.5g) were added and the slurry was stirred for 20 min. The aluminium saltswere separated by vacuum filtering the slurry through a Celite pad. Thesalts were repeatedly washed with methylene chloride. The filtrate wasdried (Na₂SO₄) and concentrated under reduced pressure. A residue waschromatographed on silica gel with hexane/ethyl acetate (90:10) toafford the alcohol 7 (96 mg, 93% yield) as a colorless oil:[α]_(D)+25.5° (c 1.0, CHCl₃); ¹HNMR (400 MHz, CDCl₃) δ 3.99 (1H, d,J=2.1 Hz, 8α-H), 3.64 (2H, t, J=6.6 Hz, CH₂OH), 1.92 (1H, dm, J=12.3Hz), 0.907 (3H, s, 18-H₃), 0.886 (9H, s, Si-t-Bu), 0.81 (3H, d, J=6.6Hz, 21-H₃), 0.007 and −0.006 (each, 3H, each s, SiMe₂); ¹³C NMR (100MHz) δ 69.43 (d, C-8), 63.18 (t, C-25), 56.31 (d), 53.10 (d), 42.17 (s,C-13), 40.65 (t), 35.05 (t), 34.70 (d), 34.45 (t), 33.20 (t), 27.17 (t),25.79 (q, SiCMe₃), 22.94 (t), 22.35 (t), 18.53 (q, C-21), 18.02 (s,SiCMe₃), 17.71 (t), 14.03 (q, C-18), −4.81 and −5.17 (each q, eachSiMe); MS (EI) m/z no M⁺, 325 (3, M⁺-C₃H₇), 311 (9, M⁺-C₄H₉), 269 (6,M⁺-C₆H₁₁O) 251 (16, M⁺-H-t-BuSiMe₂H), 235 (25, M⁺-H-t-BuSiMe₂OH), 219(29), 163 (46), 135 (78), 109 (62), 75 (100); exact mass calculated forC₁₈H₃₅O₂Si (M⁺-C₄H₉) 311.2406, found 311.2397.

Preparation of(20S)-de-A,B-8β-(tert-butyldimethylsilyl)oxy-20-butyl-pregnane (8)

To a stirred solution of the alcohol 7 (95 mg, 0.26 mmol),4-dimethylaminopyridine (5 mg, 0.04 mmol) and triethylamine (145 μL, 105mg, 1.04 mmol) in anhydrous methylene chloride (5 mL) p-toluenesulfonylchloride (68 mg, 0.36 mmol) was added at 0° C. A cooling bath wasremoved and stirring was continued for 22 h. Methylene chloride (20 mL)was added and the mixture washed with a saturated aq. NaHCO₃ solution,dried (Na₂SO₄) and concentrated under reduced pressure. A residue wasdissolved in anhydrous THF (5 mL) and lithium aluminium hydride (32 mg,0.84 mmol) was added to the solution at 0° C. A cooling bath was removedand the mixture was stirred for 18 h at room temperature. The excesshydride was quenched by careful, successive addition of sat. aq. NH₄Cl.Methylene chloride (15 mL) and Celite (0.5 g) were added and the slurrywas stirred for 20 min. The aluminium salts were separated by vacuumfiltering the slurry through a Celite pad. The salts were repeatedlywashed with methylene chloride. The filtrate was dried (Na₂SO₄) andconcentrated under reduced pressure. A residue was chromatographed onsilica gel with hexane/ethyl acetate (97:3) to give the product 8 (85mg, 93% yield): [α]_(D)+25.3° (c 1.26, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ4.00 (1H, d, J=2.1 Hz, 8α-H), 1.95 (1H, dm, J=12.4 Hz), 0.914 (3H, s,18-H₃), 0.893 (9H, s, Si-t-Bu), 0.81 (3H, d, J=6.6 Hz, 21-H₃), 0.013 and0.000 (each 3H, each s, each SiMe); ¹³C NMR (100 MHz) δ 69.52 (d, C-8),56.47 (d), 53.15 (d), 42.19 (s, C-13), 40.68 (t), 35.02 (t), 34.79 (d),34.52 (t), 28.56 (t), 27.21 (t), 25.81 (q, SiCMe₃), 23.09 (t), 22.99(t), 18.62 (q, C-21), 18.05 (s, SiCMe₃), 17.75 (t), 14.26 (q, C-25),14.02 (q, C-18), −4.79 and −5.16 (each q, each SiMe); MS (EI) m/z 352(2, M⁺), 337 (4, M⁺-CH₃), 295 (81, M⁺-t-Bu), 253 (13, M⁺-C₆H₁₁O), 219(71, M⁺-H-t-BuSiMe₂OH), 177 (10), 135 (22), 75 (100); exact masscalculated for C₁₈H₃₅OSi (M⁺-C₄H₉) 295.2457, found 295.2454.

Preparation of (20S)-de-A,B-20-butyl-pregnan-8β-ol (9)

The protected alcohol 8 (84 mg, 0.24 mmol) was dissolved in anhydrousTHF (5 mL) and anhydrous methanol (5 mL). Hydrogen fluoride-pyridinecomplex (4 mL) was added followed at room temperature and the mixturewas sired for 19 h. Ethyl acetate (20 mL) was added and the organicphase washed with brine and water, dried (Na₂SO₄) and concentrated underreduced pressure. The residue was diluted with hexane andchromatographed on silica gel with hexane to give the product 9 (17 mg,30% yield) as a colorless oil: ¹H NMR (500 MHz, CDCl₃) δ 4.07 (1H, d,J=2.5 Hz, 8α-H), 1.98 (1H, dm, J=13.1 Hz), 1.88-1.76 (3H, m), 0.927 (3H,s, 18-H₃), 0.89 (3H, t, J=7.1 Hz, 25-H₃), 0.81 (3H, d, J=6.6 Hz, 21-H₃);¹³C NMR (125 MHz) δ 69.46 (d, C-8), 56.32 (d), 52.67 (d), 41.90 (s,C-13), 40.32 (t), 34.97 (t), 34.76 (d), 33.59 (t), 28.52 (t), 27.05 (t),23.08 (t), 22.42 (t), 18.56 (q, C-21), 17.49 (t), 14.23 (q, C-25), 13.77(q, C-18).

Preparation of (20S)-de-A,B-20-butyl-pregnan-8-one (II)

Pyridinium dichromate (118 mg, 314 μmol) was added to a solution of thealcohol 9 (15 mg, 63 μmol) and pyridinium p-toluenesulfonate (2 mg, 8μmol) in anhydrous methylene chloride (5 mL). The resulting suspensionwas stirred at room temperature for 2 h. The reaction mixture wasfiltered through a Waters silica Sep-Pak cartridge (5 g) that wasfurther washed with hexane/ethyl acetate (95:5). After removal ofsolvents the ketone II (12 mg, 81% yield) was obtained as a colorlessoil: ¹H NMR (400 MHz, CDCl₃) δ 2.45 (1H, dd, J=11.5, 7.6 Hz), 2.32-2.16(2H, m), 0.90 (3H, t, J=6.9 Hz, 25-H₃), 0.85 (3H, d, J=6.1 Hz, 21-H₃),0.634 (3H, s, 18-H₃); ¹³C NMR (100 MHz) δ 212.14 (C-8), 62.01 (C-14),56.24, 49.96 (C-13), 40.96, 38.86, 35.18, 34.87, 28.43, 27.15, 24.06,23.03, 18.94 (C-21), 18.51, 14.19 (C-25), 12.72 (C-18).

Preparation of (20S)-2-methylene-19,26,27-trinor-1α-hydroxycalciferol(1)

To a solution of phosphine oxide 11 (60 mg, 103 μmol) in anhydrous THF(600 μL) at −20° C. was slowly added PhLi (1.8 M in cyclohexane-ether,60 μL, 108 μmol) under argon with stirring. The solution turned deeporange. After 30 min the mixture was cooled to −78° C. and a precooled(−78° C.) solution of ketone II (12 mg, 51 μmol) in anhydrous THF (200μL) was slowly added. The mixture was stirred under argon at −78° C. for3 h and at 0° C. for 18 h. Ethyl acetate was added, and the organicphase was washed with brine, dried (Na₂SO₄) and evaporated. The residuewas dissolved in hexane and applied on a Waters silica Sep-Pak cartridge(2 g). The cartridge washed with hexane and hexane/ethyl acetate(99.5:0.5) to give 19-norvitamin derivative 12 (13 mg). The Sep-Pak wasthen washed with hexane/ethyl acetate (96:4) to recover the unchangedC,D-ring ketone II (6 mg, 25 μmol), and with ethyl acetate to recoverdiphenylphosphine oxide 11 (56 mg). The protected vitamin 12 was furtherpurified by HPLC (9.4×250 mm Zorbax-Silica column, 4 mL/min) usinghexane/2-propanol (99.9:0.1) solvent system. Pure compound 12 (8.3 mg,53% yield) was eluted at R_(t)=3.2 min as a colorless oil: MS (EI) m/z600 (14, M⁺), 585 (4, M⁺-Me), 543 (11, M⁺-C₄H₉), 468 (100,M⁺-t-BuMe₂SiOH), 366 (43), 323 (9), 257 (13), 234 (16), 147 (24), 73(97); exact mass calculated for C₃₇H₆₈O₂Si₂ (M⁺) 600.4758, found600.4742.

Protected vitamin 12 (8 mg, 13 μmol) was dissolved in anhydrous THF (4mL) and a solution of tetrabutylammonium fluoride (1 M in THF, 100 μL,100 μmol) was added, followed by freshly activated molecular sieves 4A(300 mg). The mixture was stirred under argon at room temperature for 4h, then diluted with 2 mL of hexane/ethyl acetate (9:1) and applied on aWaters silica Sep-Pak cartridge (2 g). Elution with the same solventsystem gave the crude product I that was further purified by HPLC(9.4×250 mm Zorbax-Silica column, 4 mL/min) using hexane/2-propanol(9:1) solvent system. Analytically pure 2-methylene-19-norvitamin I(3.59 mg, 74% yield) was collected at R_(t)=6.4 min. as a colorless oil:UV (in EtOH) λ_(max) 261, 251, 243 nm; ¹H NMR (750 MHz, CDCl₃) δ 6.36and 5.89 (1H and 1H, each d, J=11.2 Hz, 6- and 7-H), 5.11 and 5.09 (each1H, each s, ═CH₂), 4.47 (2H, m, 1β- and 30α-H), 2.84 (1H, dd, J=13.3,4.4 Hz, 10β-H), 2.82 (1H, br d, J=12.3 Hz, 9β-H), 2.58 (1H, dd, J=13.3,3.4 Hz, 4α-H), 2.32 (1H, dd, J=13.3, 6.1 Hz, 4β-H), 2.30 (1H, dd,J=13.3, 8.4 Hz, 10α-H), 2.05-1.95 (2H, m), 1.90-1.82 (1H, m), 0.89 (3H,t, J=7.1 Hz, 25-H₃), 0.84 (3H, d, J=6.5 Hz, 21-H₃), 0.552 (3H, s,18-H₃); ¹³C NMR (100 MHz) δ 151.97 (s, C-2), 143.55 (s, C-8), 130.29 (s,C-5), 124.30 (d, C-6), 115.24 (d, C-7), 107.71 (t, ═CH₂), 71.82 and70.70 (each d, C-1 and C-3), 56.36 (d), 56.22 (d), 45.84 (s, C-13),45.79 (t), 40.34 (t), 38.16 (t), 35.45 (d), 35.29 (t), 28.98 (t), 28.55(t), 27.29 (t), 23.51 (t), 23.08 (t), 22.17 (t), 18.59 (q, C-21), 14.23(q, C-25), 12.33 (q, C-18); MS (EI) m/z 372 (100, M⁺), 354 (4, M⁺-H₂O),324 (15, M⁺-H₂O—C₂H₆), 287 (60, M⁺-C₆H₁₃), 269 (22, M⁺-C₆H₁₃—H₂O), 251(18, M⁺-C₆H₁₃-2H₂O), 231 (22), 219 (35), 147 (46), 135(76), 119 (27),107 (61); exact mass calculated for C₂₅H₄₀O₂ (M⁺) 372.3028 found372.3039.

Biological Activity of2-Methylene-19,26,27-Trinor-(20S)-1α-Hydroxyvitamin D₃

The introduction of a methylene group to the 2-position and the removalof the two methyl groups at the 26 and 27 positions in the side chain of1α-hydroxy-19-nor-(20S) -vitamin D₃ had little or no effect on bindingto the full length recombinant rat vitamin D receptor, as compared to1α,25-dihydroxyvitamin D₃. The compound OM bound equally well to thereceptor as compared to the standard 1,25-(OH)₂D₃ (FIG. 1). It might beexpected from these results that compound OM would have equivalentbiological activity. Surprisingly, however, compound OM is a highlyselective analog with unique biological activity.

FIG. 6 shows that OM has very little activity as compared to that of1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), the natural hormone, instimulating intestinal calcium transport.

FIGS. 4 and 5 demonstrate that OM has very little bone calciummobilization activity, as compared to 1,25(OH)₂D₃.

FIGS. 4-6 thus illustrate that OM may be characterized as having little,if any, calcemic activity.

FIG. 2 illustrates that OM is almost as potent as 1,25(OH)₂D₃ on HL-60cell differentiation, making it an excellent candidate for the treatmentof psoriasis and cancer, especially against leukemia, colon cancer,breast cancer, skin cancer and prostate cancer. In addition, due to itsrelatively high cell differentiation activity, this compound provides atherapeutic agent for the treatment of various skin conditions includingwrinkles, lack of adequate dermal hydration, i.e. dry skin, lack ofadequate skin firmness, i.e. slack skin, and insufficient sebumsecretion. Use of this compound thus not only results in moisturizing ofskin but also improves the barrier function of skin.

FIG. 3 illustrates that the compound OM has transcriptional activity inbone cells, but less than 1α,25-dihydroxyvitamin D₃. This result,together with the cell differentiation activity of FIG. 2, suggests thatOM will be very effective in psoriasis because it has direct cellularactivity in causing cell differentiation and in suppressing cell growth.These data also indicate that OM may have significant activity as ananti-cancer agent, especially against leukemia, colon cancer, breastcancer, skin cancer and prostate cancer. The strong activity of OM onHL-60 differentiation suggests it will be active in suppressing growthof parathyroid glands and in the suppression of the preproparathyroidgene.

Experimental Methods

Vitamin D Receptor Binding

Test Material

Protein Source

Full-length recombinant rat receptor was expressed in E. coli BL21 (DE3)Codon Plus RIL cells and purified to homogeneity using two differentcolumn chromatography systems. The first system was a nickel affinityresin that utilizes the C-terminal histidine tag on this protein. Theprotein that was eluted from this resin was further purified using ionexchange chromatography (S-Sepharose Fast Flow). Aliquots of thepurified protein were quick frozen in liquid nitrogen and stored at −80°C. until use. For use in binding assays, the protein was diluted inTEDK₅₀ (50 mM Tris, 1.5 mM EDTA, pH7.4, 5 mM DTT, 150 mM KCl) with 0.1%Chaps detergent. The receptor protein and ligand concentration wereoptimized such that no more than 20% of the added radiolabeled ligandwas bound to the receptor.

Study Drugs

Unlabeled ligands were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry (1,25(OH)₂D₃: molar extinctioncoefficient=18,200 and λ_(max)=265 nm; Analogs: molar extinctioncoefficient=42,000 and λ_(max)=252 nm). Radiolabeled ligand(³H-1,25(OH)₂D₃, ˜159 Ci/mmole) was added in ethanol at a finalconcentration of 1 nM.

Assay Conditions

Radiolabeled and unlabeled ligands were added to 100 mcl of the dilutedprotein at a final ethanol concentration of ≦10%, mixed and incubatedovernight on ice to reach binding equilibrium. The following day, 100mcl of hydroxylapatite slurry (50%) was added to each tube and mixed at10-minute intervals for 30 minutes. The hydroxylapaptite was collectedby centrifugation and then washed three times with Tris-EDTA buffer (50mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Titron X-100. After thefinal wash, the pellets were transferred to scintillation vialscontaining 4 ml of Biosafe II scintillation cocktail, mixed and placedin a scintillation counter. Total binding was determined from the tubescontaining only radiolabeled ligand.

HL-60 Differentiation

Test Material

Study Drugs

The study drugs were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry. Serial dilutions were prepared sothat a range of drug concentrations could be tested without changing thefinal concentration of ethanol (≦0.2%) present in the cell cultures.

Cells

Human promyelocytic leukemia (HL60) cells were grown in RPMI-1640 mediumcontaining 10% fetal bovine serum. The cells were incubated at 37° C. inthe presence of 5% CO₂.

Assay Conditions

HL60 cells were plated at 1.2×10⁵ cells/ml. Eighteen hours afterplating, cells in duplicate were treated with drug. Four days later, thecells were harvested and a nitro blue tetrazolium reduction assay wasperformed (Collins et al., 1979; J. Exp. Med. 149:969-974). Thepercentage of differentiated cells was determined by counting a total of200 cells and recording the number that contained intracellularblack-blue formazan deposits. Verification of differentiation tomonocytic cells was determined by measuring phagocytic activity (datanot shown).

In Vitro Transcription Assay

Transcription activity was measured in ROS 17/2.8 (bone) cells that werestably transfected with a 24-hydroxylase (24Ohase) gene promoterupstream of a luciferase reporter gene (Arbour et al., 1998). Cells weregiven a range of doses. Sixteen hours after dosing the cells wereharvested and luciferase activities were measured using a luminometer.

RLU=relative luciferase units.

Intestinal Calcium Transport and Bone Calcium Mobilization

Male, weanling Sprague-Dawley rats were placed on Diet 11 (0.47% Ca)diet +AEK for one week followed by Diet 11 (0.02% Ca) +AEK for 3 weeks.The rats were then switched to a diet containing 0.47% Ca for one weekfollowed by two weeks on a diet containing 0.02% Ca. Dose administrationbegan during the last week on 0.02% calcium diet. Four consecutive ipdoses were given approximately 24 hours apart. Twenty-four hours afterthe last dose, blood was collected from the severed neck and theconcentration of serum calcium determined as a measure of bone calciummobilization. The first 10 cm of the intestine was also collected forintestinal calcium transport analysis using the everted gut sac method.

Interpretation of Data

VDR binding, HL60 cell differentiation, and transcription activity. OM(K_(i)=7.7×10⁻¹¹M) is nearly equivalent to the natural hormone1α,25-dihydroxyvitamin D₃ (K_(i)=4.4×10¹¹M) in its ability to competewith [³H]-1,25(OH)₂D₃ for binding to the full-length recombinant ratvitamin D receptor (FIG. 1). There is also little difference between OM(EC₅₀=4.7×10⁻⁹M) in its ability (efficacy or potency) to promote HL60differentiation as compared to 1α,25-dihydroxyvitamin D₃(EC₅₀=2.9×10⁻⁹M) (See FIG. 2). Compound OM (EC₅₀=3.2×10⁻⁹M) hastranscriptional activity in bone cells but less than1α,25-dihydroxyvitamin D₃ (EC₅₀=1.9×10⁻¹⁰M) (See FIG. 3). These resultssuggest that OM will be very effective in psoriasis because it hasdirect cellular activity in causing cell differentiation and insuppressing cell growth. These data also indicate that OM will havesignificant activity as an anti-cancer agent, especially againstleukemia, colon cancer, breast cancer, skin cancer and prostate cancer,as well as against skin conditions such as dry skin (lack of dermalhydration), undue skin slackness (insufficient skin firmness),insufficient sebum secretion and wrinkles. It would also be expected tobe very active in suppressing secondary hyperparathyroidism.

Calcium mobilization from bone and intestinal calcium absorption invitamin D-deficient animals. Using vitamin D-deficient rats on a lowcalcium diet (0.02%), the activities of OM and 1,25(OH)₂D₃ in intestineand bone were tested. As expected, the native hormone (1,25(OH)₂D₃)increased serum calcium levels at all dosages (FIG. 4). FIG. 4 and FIG.5 show that OM has little, if any, activity in mobilizing calcium frombone. Administration of OM at 87 pmol/day for 4 consecutive days did notresult in mobilization of bone calcium, and increasing the amount of OMto 780 pmol/day and then to 7020 pmol/day was also without anysubstantial effect.

Intestinal calcium transport was evaluated in the same groups of animalsusing the everted gut sac method (FIG. 6). These results show that thecompound OM does not promote intestinal calcium transport whenadministered at either 87 or 780 pmol/day, whereas 1,25(OH)₂D₃ promotesa significant increase at the 87 and 780 pmol/day doses. It was onlywhen 7020 pmol/day of OM was administered that significant intestinalcalcium transport activity was recorded, an almost 10-fold increase indosage over the 780 pmol/day dose. Thus, it may be concluded that OM isessentially devoid of intestinal calcium transport activity at therecommended lower doses.

These results illustrate that OM is an excellent candidate for numeroushuman therapies as described herein, and that it may be particularlyuseful in a number of circumstances such as suppression of secondaryhyperparathyroidism of renal osteodystrophy, autoimmune diseases,cancer, and psoriasis. OM is an excellent candidate for treatingpsoriasis because: (1) it has significant VDR binding, transcriptionactivity and cellular differentiation activity; (2) it is devoid ofhypercalcemic liability unlike 1,25(OH)₂D₃; and (3) it is easilysynthesized. Since OM has significant binding activity to the vitamin Dreceptor, but has little ability to raise blood serum calcium, it mayalso be particularly useful for the treatment of secondaryhyperparathyroidism of renal osteodystrophy.

These data also indicate that the compound OM of the invention may beespecially suited for treatment and prophylaxis of human disorders whichare characterized by an imbalance in the immune system, e.g. inautoimmune diseases, including multiple sclerosis, lupus, diabetesmellitus, host versus graft rejection, and rejection of organtransplants; and additionally for the treatment of inflammatorydiseases, such as rheumatoid arthritis, asthma, and inflammatory boweldiseases such as celiac disease, ulcerative colitis and Crohn's disease.Acne, alopecia and hypertension are other conditions which may betreated with the compound OM of the invention.

The compounds of the invention of formula I, and particularly formulaIa, are also useful in preventing or treating obesity, inhibitingadipocyte differentiation, inhibiting SCD-1 gene transcription, and/orreducing body fat in animal subjects. Therefore, in some embodiments, amethod of preventing or treating obesity, inhibiting adipocytedifferentiation, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal subject includes administering to the animalsubject, an effective amount of one or more of the compounds or apharmaceutical composition that includes one or more of the compounds offormula I. Administration of the compound or the pharmaceuticalcompositions to the subject inhibits adipocyte differentiation, inhibitsgene transcription, and/or reduces body fat in the animal subject. Theanimal may be a human, a domestic animal such as a dog or a cat, or anagricultural animal, especially those that provide meat for humanconsumption, such as fowl like chickens, turkeys, pheasant or quail, aswell as bovine, ovine, caprine, or porcine animals.

For prevention and/or treatment purposes, the compounds of thisinvention defined by formula I may be formulated for pharmaceuticalapplications as a solution in innocuous solvents, or as an emulsion,suspension or dispersion in suitable solvents or carriers, or as pills,tablets or capsules, together with solid carriers, according toconventional methods known in the art. Any such formulations may alsocontain other pharmaceutically-acceptable and non-toxic excipients suchas stabilizers, anti-oxidants, binders, coloring agents or emulsifyingor taste-modifying agents.

The compounds of formula I, and particularly OM, may be administeredorally, topically, parenterally, rectally, nasally, sublingually ortransdermally. The compounds may be advantageously administered byinjection or by intravenous infusion or suitable sterile solutions, orin the form of liquid or solid doses via the alimentary canal, or in theform of creams, ointments, patches, or similar vehicles suitable fortransdermal applications. A dose of from 0.01 μg to 1000 μg per day ofthe compounds I, and particularly compound OM, preferably from about 0.1μg to about 500 μg per day, is appropriate for prevention and/ortreatment purposes, such dose being adjusted according to the disease tobe treated, its severity and the response of the subject as is wellunderstood in the art. Since the compound exhibits specificity ofaction, each may be suitably administered alone, or together with gradeddoses of another active vitamin D compound—e.g. 1α-hydroxyvitamin D₂ orD₃, or 1α,25-dihydroxyvitamin D₃—in situations where different degreesof bone mineral mobilization and calcium transport stimulation is foundto be advantageous.

Compositions for use in the above-mentioned treatments comprise aneffective amount of the compounds, particularly the compound OM asdefined by the above formula I and Ia as the active ingredient, and asuitable carrier. An effective amount of such compound for use inaccordance with this invention is from about 0.01 μg to about 1000 μgper gm of composition, preferably from about 0.1 μg to about 500 μg pergram of composition, and may be administered topically, transdermally,orally, rectally, nasally, sublingually or parenterally in dosages offrom about 0.01 μg/day to about 1000 μg/day, and preferably from about0.1 μg/day to about 500 μg/day.

The compounds I, and particularly compound OM may be formulated ascreams, lotions, ointments, topical patches, pills, capsules or tablets,suppositories, aerosols, or in liquid form as solutions, emulsions,dispersions, or suspensions in pharmaceutically innocuous and acceptablesolvent or oils, and such preparations may contain in addition otherpharmaceutically innocuous or beneficial components, such asstabilizers, antioxidants, emulsifiers, coloring agents, binders ortaste-modifying agents.

The compounds I, and particularly compound OM is advantageouslyadministered in amounts sufficient to effect the differentiation ofpromyelocytes to normal macrophages. Dosages as described above aresuitable, it being understood that the amounts given are to be adjustedin accordance with the severity of the disease, and the condition andresponse of the subject as is well understood in the art.

The formulations of the present invention comprise an active ingredientin association with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredients. The carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion.

Formulations for rectal administration may be in the form of asuppository incorporating the active ingredient and carrier such ascocoa butter, or in the form of an enema.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

Formulations suitable for topical administration include liquid orsemi-liquid preparations such as liniments, lotions, applicants,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes; or solutions or suspensions such as drops; or as sprays.

For nasal administration, inhalation of powder, self-propelling or sprayformulations, dispensed with a spray can, a nebulizer or an atomizer canbe used. The formulations, when dispensed, preferably have a particlesize in the range of 10 to 100μ.

The formulations may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.By the term “dosage unit” is meant a unitary, i.e. a single dose whichis capable of being administered to a patient as a physically andchemically stable unit dose comprising either the active ingredient assuch or a mixture of it with solid or liquid pharmaceutical diluents orcarriers.

1. A compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 2. The compound of claim 1wherein X₂ is hydrogen.
 3. The compound of claim 1 wherein X₁ ishydrogen.
 4. The compound of claim 1 wherein X₁ and X₂ are botht-butyldimethylsilyl.
 5. A pharmaceutical composition containing aneffective amount of at least one compound as claimed in claim 1 togetherwith a pharmaceutically acceptable excipient.
 6. The pharmaceuticalcomposition of claim 5 wherein said effective amount comprises fromabout 0.01 μg to about 1000 μg per gram of composition.
 7. Thepharmaceutical composition of claim 5 wherein said effective amountcomprises from about 0.1 μg to about 500 μg per gram of composition. 8.2-methylene-19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃ having theformula:


9. A pharmaceutical composition containing an effective amount of2-methylene-19,26,27-trinor-(20 S)-1α-hydroxyvitamin D₃ together with apharmaceutically acceptable excipient.
 10. The pharmaceuticalcomposition of claim 9 wherein said effective amount comprises fromabout 0.01 μg to about 1000 μg per gram of composition.
 11. Thepharmaceutical composition of claim 9 wherein said effective amountcomprises from about 0.1 μg to about 500 μg per gram of composition. 12.A method of treating psoriasis comprising administering to a subjectwith psoriasis an effective amount of a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 13. The method of claim 12wherein the compound is administered orally.
 14. The method of claim 12wherein the compound is administered parenterally.
 15. The method ofclaim 12 wherein the compound is administered transdermally.
 16. Themethod of claim 12 wherein the compound is administered topically. 17.The method of claim 12 wherein the compound is administered rectally.18. The method of claim 12 wherein the compound is administered nasally.19. The method of claim 12 wherein the compound is administeredsublingually.
 20. The method of claim 12 wherein the compound isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.21. The method of claim 12 wherein the compound is2-methylene-19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃ having theformula:


22. A method of treating a disease selected from the group consisting ofleukemia, colon cancer, breast cancer, skin cancer or prostate cancercomprising administering to a subject with said disease an effectiveamount of a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 23. The method of claim 22wherein the compound is administered orally.
 24. The method of claim 22wherein the compound is administered parenterally.
 25. The method ofclaim 22 wherein the compound is administered transdermally.
 26. Themethod of claim 22 wherein the compound is administered rectally. 27.The method of claim 22 wherein the compound is administered nasally. 28.The method of claim 22 wherein the compound is administeredsublingually.
 29. The method of claim 22 the compound is administered ina dosage of from about 0.01 μg/day to about 1000 μg/day.
 30. The methodof claim 22 wherein the compound is2-methylene-19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃ having theformula:


31. A method of treating an autoimmune disease selected from the groupconsisting of multiple sclerosis, lupus, diabetes mellitus, host versusgraft rejection, and rejection of organ transplants, comprisingadministering to a subject with said disease an effective amount of acompound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 32. The method of claim 31wherein the compound is administered orally.
 33. The method of claim 31wherein the compound is administered parenterally.
 34. The method ofclaim 31 wherein the compound is administered transdermally.
 35. Themethod of claim 31 wherein the compound is administered rectally. 36.The method of claim 31 wherein the compound is administered nasally. 37.The method of claim 31 wherein the compound is administeredsublingually.
 38. The method of claim 31 wherein the compound isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.39. The method of claim 31 wherein the compound is2-methylene-19,26,27-trinor-(20 S)-1α-hydroxyvitamin D₃ having theformula:


40. A method of treating an inflammatory disease selected from the groupconsisting of rheumatoid arthritis, asthma, and inflammatory boweldiseases, comprising administering to a subject with said disease aneffective amount of a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 41. The method of claim 40wherein the compound is administered orally.
 42. The method of claim 40wherein the compound is administered parenterally.
 43. The method ofclaim 40 wherein the compound is administered transdermally.
 44. Themethod of claim 40 wherein the compound is administered rectally. 45.The method of claim 40 wherein the compound is administered nasally. 46.The method of claim 40 wherein the compound is administeredsublingually.
 47. The method of claim 40 wherein the compound isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.48. The method of claim 40 wherein the compound is2-methylene-19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃ having theformula:


49. A method of treating a skin condition selected from the groupconsisting of wrinkles, lack of adequate skin firmness, lack of adequatedermal hydration and insufficient sebum secretion which comprisesadministering to a subject with said skin condition an effective amountof a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 50. The method of claim 49wherein the compound is administered orally.
 51. The method of claim 49wherein the compound is administered parenterally.
 52. The method ofclaim 49 wherein the compound is administered transdermally.
 53. Themethod of claim 49 wherein the compound is administered topically. 54.The method of claim 49 wherein the compound is administered rectally.55. The method of claim 49 wherein the compound is administered nasally.56. The method of claim 49 wherein the compound is administeredsublingually.
 57. The method of claim 49 wherein the compound isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.58. The method of claim 49 wherein the compound is2-methylene-19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃ having theformula:


59. A method of treating renal osteodystrophy comprising administeringto a subject with renal osteodystrophy an effective amount of thecompound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 60. The method of claim 59wherein the compound is administered orally.
 61. The method of claim 59wherein the compound is administered parenterally.
 62. The method ofclaim 59 wherein the compound is administered transdermally.
 63. Themethod of claim 59 wherein the compound is administered rectally. 64.The method of claim 59 wherein the compound is administered nasally. 65.The method of claim 59 wherein the compound is administeredsublingually.
 66. The method of claim 59 wherein the compound isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.67. The method of claim 59 wherein the compound is2-methylene-19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃ having theformula:


68. A method of treating or preventing obesity of an animal, inhibitingadipoctye differentiation, inhibiting SCD-1 gene transcription, and/orreducing body fat in an animal comprising administering to an animal inneed thereof an effective amount of a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 69. The method of claim 68wherein the compound is administered orally.
 70. The method of claim 68wherein the compound is administered parenterally.
 71. The method ofclaim 68 wherein the compound is administered transdermally.
 72. Themethod of claim 68 wherein the compound is administered rectally. 73.The method of claim 68 wherein the compound is administered nasally. 74.The method of claim 68 wherein the compound is administeredsublingually.
 75. The method of claim 68 wherein the compound isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.76. The method of claim 68 wherein the compound is2-methylene-19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃ having theformula:


77. The method of claim 68 wherein the animal is a human.
 78. The methodof claim 68 wherein the animal is a domestic animal.
 79. The method ofclaim 68 wherein the animal is an agricultural animal.
 80. A compoundhaving the formula: